Volume 19, No 9, Sep 2009

ISSN: 1001-0602 
EISSN: 1748-7838 2018 
impact factor 17.848* 
(Clarivate Analytics, 2019)

Volume 19 Issue 9, September 2009: 1098-1109

ORIGINAL ARTICLES

A unique sequence in the N-terminal regulatory region controls the nuclear localization of KLF8 by cooperating with the C-terminal zinc-fingers

Tina S Mehta, Heng Lu, Xianhui Wang, Alison M Urvalek, Kim-Hang H Nguyen, Farah Monzur, Jojo D Hammond, Jameson Q Ma and Jihe Zhao

Correspondence: Jihe Zhao,(zhaojh@mail.amc.edu)

Krüppel-like factor 8 (KLF8) transcription factor plays a critical role in cell cycle progression, oncogenic transformation, epithelial to mesenchymal transition and invasion. However, its nuclear localization signal(s) (NLS) has not been identified. KLF8 shares with other KLFs monopartite NLSs (mNLS) and C₂H₂ zinc fingers (ZFs), both of which have been shown to be the NLSs for some other KLFs. In this report, using PCR-directed mutagenesis and immunofluorescent microscopy, we show that disruption of the mNLSs, deletion of any single ZF, or mutation of the Zn²⁺-binding or DNA-contacting motifs did not affect the nuclear localization of KLF8. Deletion of >1.5 ZFs from C-terminus, however, caused cytoplasmic accumulation of KLF8. Surprisingly, deletion of amino acid (aa) 151-200 region almost eliminated KLF8 from the nucleus. S165A, K171E or K171R mutation, or treatment with PKC inhibitor led to partial cytoplasmic accumulation. Co-immunoprecipitation demonstrated that KLF8 interacted with importin-β and this interaction required the ZF motif. Deletion of aa 1-150 or 201-261 region alone did not alter the nuclear localization. BrdU incorporation and cyclin D1 promoter luciferase assays showed that the KLF8 mutants defective in nuclear localization could not promote DNA synthesis or cyclin D1 promoter activation as the wild-type KLF8 did. Taken together, these results suggest that KLF8 has two NLSs, one surrounding S165 and K171 and the other being two tandem ZFs, which are critical for the regulation of KLF8 nuclear localization and its cellular functions.

Cell Research (2009) 19:1098-1109. doi: 10.1038/cr.2009.64 published online 2 June 2009

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